Title

Author

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Burton, Jeremy P.

2nd Supervisor

Reid, Gregor

Co-Supervisor

Abstract

There has been a growing interest in human microbiome studies in the past decade, with the development of high-throughput sequencing techniques. These microorganisms interact and respond to the host as an entity, and are involved in various homeostatic functions including nutrition digestion, immune response, metabolism and endocrine regulation. The urinary microbiome, however, remains relatively under-investigated.

One of the technical challenges of urinary microbiome studies is the samples usually contain a large number of host cells and low microbial biomass. These samples with the high host, low microbial abundance (“high-low” samples) are associated with increased risk of compromised quality of 16s rRNA gene sequencing results. An analysis with mock samples showed that mechanisms of host materials interfering with microbiome analysis includes reducing microbial DNA extract yield by competitively binding to the filter of DNA extraction column, inhibiting PCR amplification of 16S rRNA gene regions as non-target DNA, and consuming sequencing depth by unspecific amplification from PCR. To counter these issues, a refined processing protocol and a quality checking tool were developed for handling “high-low” samples. With these methods, a combination of sequencing-based methods and enhanced culture-based methods showed evidence of bacteria in renal tissue samples.

On the other hand, the optimal urine sample collection and storage methods for microbiome study have not been reported. An optimisation experiment showed that urine samples with a volume higher than 20 mL and stored in centrifuged pellets generated the best sequencing results.

The urinary microbiome of healthy subjects and urinary stone patients were characterised using 16s rRNA gene sequencing and enhanced quantitative urine culture (EQUC) techniques. Although no clear distinction was observed of urinary microbiome profiles between healthy subjects and urinary stone patients, male and female individuals do have their unique urinary microbiome profiles. The urinary microbiome profile of an individual remained stable throughout three months.

Investigation of urine samples of metabolic stone patients before and after lithotripsy showed fluctuations in their urinary microbiome profiles, with newly-emerged microbes in sequencing results correlated with microbes cultured from stone samples. These results suggested bacteria liberated from metabolic stones during lithotripsy.